Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

Summary

Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.

Abstract

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

Introduction

Fluorescence microscopy using antibodies targeting specific proteins is routinely used to visualize proteins of interest in cell culture and tissues. A major complication to the acquisition of clear and definitive images in immunofluorescence is autofluorescence, which can be caused endogenously in mammalian tissue by the age pigment lipofuscin and by proteins such as elastin and collagen^1,2. Other sources of autofluorescence can be introduced through sample preparation steps such as aldehyde fixation³. Lipofuscin granules, composed primarily of oxidatively modified protein and lipid de....

Protocol

Note: The work presented was performed in compliance with recognized international standards, including the International Conference on Harmonization (ICH), the Council for International Organizations of Medical Sciences (CIOMS), and the principles of the Declaration of Helsinki. Use of human tissue was with the approval of University Health Network Research Ethics Board. The human brain samples were collected as a part of the Maritime Brain Tissue Bank. At the time of collection, informed consent was obtained from all patients.

1. Construction of photobleaching apparatus and solutions

1. Prepare stock solutions.
   1. Prepar....

Results

The photobleaching pre-treatment step can be added to a standard immunofluorescence protocol immediately prior to antigen retrieval and immunostaining (Figure 1A). Assembly of the photobleaching apparatus can also be performed using various, inexpensive, off-the-shelf components (Figure 1B). The emission spectrum of white phosphor LEDs covers a wide range of wavelengths which makes them suitable for broad-range photobleaching, ag.......

Discussion

The photobleaching pre-treatment of tissues described in this manuscript allows for effective elimination of autofluorescence using off-the-shelf components. The protocol describes immunofluorescence imaging of phosphorylated tau aggregates in formalin-fixed human brain tissue using secondary antibodies conjugated to Alexa 488 and Texas Red, with DAPI as a nuclear counterstain. To apply the method to other tissues, we recommend performing a 48 h photobleaching pre-treatment to the sample as a starting point. After photob.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trizma Base	Sigma-Aldrich	T6066
Sodium Choloride	Sigma-Aldrich	S7653
Hydrochloric Acid	Caledon Laboratory Chemicals	1506656
Sodium Azide	BioShop Canada	SAZ001
100 mm x 100 mm x 20 mm Pitri dish	Sarstedt	82.9923.422	All components of photobleacher can be substituted based on availability
6 W LED Dimmable Desk Lamp	DBPower	DS501	All components of photobleacher can be substituted based on availability
Citric Acid	Sigma-Aldrich	C-2404
Ethylenediaminetetraacetic acid (EDTA)	BioShop Canada	EDT001
Tween 20	Sigma-Aldrich	P-7949
Sodium Hydroxide	BioShop Canada	SHY700.1
Water bath	Haake Fisons	K15
Slide collector	FisherScientific	12-587-17B
Staining Jar	FisherScientific	E94
Orbital Shaker	Bellco Glass	7744-08115
Triton X-100	Sigma-Aldrich	T7878
Bovine Serum Albumin	FisherScientific	BP1600-1
Normal Goat Serum	Aurion	905.002
Hydrophobic pen	Sigma-Aldrich	Z672548-1EA
Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)	ThermoFisher	MN1020
Goat anti-Mouse Secondary Antibody, Texas Red-X	ThermoFisher	T862
Goat anti-Mouse Secondary Antibody, Alexa Fluor 488	ThermoFisher	A-11029
DAPI	Sigma-Aldrich	D9542
Mounting medium	ThermoScientific	28-600-42
Glass soverslip
Confocal Microscope	Zeiss	LSM710
Imaging software ZEN 2012 Black Edition 11.0	Zeiss	LSM710	Software accompanies the Confocal Microscope
ImageJ	NIH		https://imagej.nih.gov/ij/download.html
RGB Profile Tools macro	NIH		https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt
Commercial chemical quencher	Biotum	23007